For Research Use Only. Not for use in diagnostic procedures.
Bender MedSystems GmbH | Campus Vienna Biocenter 2 | 1030 Vienna, Austria
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The information in this guide is subject to change without notice.
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ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
RevisionDateDescription
C.002 March 2021Updated the reagent volumes in the tables.
B.020 February 2020Updated manufacturing address
A.027 August 2018New document
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IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix
in this document.
Product description
How the QuantiGene™ Plex Assay works
The Invitrogen™ QuantiGene™ Plex Assay Kit enables the multiplexed measurement of gene expression
by combining branched DNA (bDNA) signal amplification with Luminex™xMAP™ multi-analyte profiling
technology. The bDNA assay is a probe hybridization-based method of target-specific RNA capture and
quantitation, amplifying signal rather than the target.
Color-coded fluorescent magnetic microspheres (capture beads) capture specific target RNA molecules
through hybridization of a custom oligonucleotide probe set, which consists of 3 types of probes:
capture extenders, label extenders, and blocking probes. The probe set hybridizes a contiguous
sequence of each target RNA. The capture extenders discriminate between dierent capture beads
based on the sequence of a capture probe conjugated to each bead, and the label extenders have tails
that provide the support for the branched DNA signal amplification.
Product information
Each amplification unit is constructed through sequential hybridization of bDNA oligonucleotides (preamplifier,amplifier, and label probe). The label probe is biotinylated to bind Streptavidin-conjugated
R-Phycoerythrin (SAPE). The resulting fluorescence signal is associated with individual capture beads
by the Luminex™ instrument, which combines advanced fluidics, optics, and digital signal processing.
Signal is reported as median fluorescence intensity (MFI) and is proportional to the number of target
RNA molecules present in the sample.
Product Description
The QuantiGene™ Plex Assay Kit consists of 3 modules, each sold separately:
•
QuantiGene™ Sample Processing Kit: contains reagents for release and stabilization of sample
RNA from cultured cells, blood (whole blood, PAXgene™ blood, Tempus™ blood, or dried blood
spots), or tissues (fresh, frozen or FFPE). These kits are not required if working with purified RNA
samples
•
QuantiGene™ Plex Assay Kit: contains the generic reagents, plates, and seals required for running
the assay
•
QuantiGene™ Plex Panel: contains the custom target-specific pooled probe set and associated
magnetic capture beads to capture user-defined genes of interest
QuantiGene
™
Plex Gene Expression Assay User Guide
5
Product information
Product description
This user guide contains instructions for using the QuantiGene™ Plex Assay with the following sample
types:
•
Cell lysates from cultured cells and whole blood
•
Tissue homogenates from fresh, frozen, or formalin-fixed,paran-embedded (FFPE) tissues
•
Purified or in vitro transcribed (IVT) RNA
For instructions on preparing cell lysates or tissue homogenates, please refer to the appropriate
QuantiGene™ Sample Processing Kit package insert.
Precautions and technical hints
•
The shaking incubator must be calibrated for both 54°C & 50°C using the Temperature Validation
Kit. The Vortemp 56 requires an inverted plate lid to be placed below the assay plates. See
instructions for temperature calibration in the Temperature Validation Kit package insert.
•
When running a new sample type, optimize input by running a dilution series to ensure that all
target signals are within the dynamic range of the assay.
•
Run samples in technical replicates. We recommend a minimum of duplicates, but ideally more in
order to calculate intra-assay precision.
•
Use fresh pipette tips when loading samples into each well. Avoid creating bubbles when pipetting.
Use a multi-channel pipette whenever possible to achieve optimal assay precision.
•
Be careful not to invert the plate or allow contents from one well to mix to another well. The
Magnetic Separation Plate is to be inverted only when removing reagents and wash buer with the
Handheld Magnetic Plate Washer.
•
On day 2 of the assay, turn on and initiate startup protocol of the Luminex™ instrument according
to the manufacturer's instructions. Lasers require 30 minutes to warm-up.
Required equipment and materials not provided
Unless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates that
the material is available from fisherscientific.com or another major laboratory supplier.
Required Equipment/Material
Handheld Magnetic Plate WasherEPX-55555-000
Microtiter plate shaker (must have 3 mm orbit at 600-800
rpm)
Vortex mixerMLS
Adjustable single and multi-channel precision pipettes
for dispensing 1-20 uL, 20-200 uL, and 200-1000uL
Reagent reservoirs (25 mL and 100 mL capacity)3054-1002 or equivalent (VistaLab
Nuclease Free Water (H2 O)MLS
Plate centrifuge capable of 240 × g speedsMLS
88880023 or 88880024, QP0706 (IKA™MS3
CLS4873 or equivalent (Corning™Costar™)
Source
Digital)
MLS
Technologies™)
6
QuantiGene™ Plex Gene Expression Assay User Guide
Product information
Contents and storage
(continued)
Required Equipment/MaterialSource
MicrocentrifugeMLS
QuantiGene™ Incubator Temperature Validation KitQS0517
4" Soft Rubber RollerQS0515
Use one of the following:
FLEXMAP 3D™instrumentAPX1342
Luminex Corporation (sold through Thermo
Fisher Scientific)
Luminex™ 200™ instrumentAPX10031
Luminex Corporation (sold through Thermo
Fisher Scientific)
MAGPIX™ instrumentLuminex Corporation
Use one of the following:
Labnet VorTemp™ 56 Shaking IncubatorQP0703 or QP0704 (include Temperature
MaxQ™ 4450 Benchtop Orbital ShakerSHKE4450 or SHKE4450-1CE (required in
Contents and storage
QuantiGene™ Plex Assay kit
The QuantiGene™ Plex Assay Kit is supplied in 3 separate boxes based on storage temperature.
Storage conditions are listed below. Refer to the product labels for expiration dating, and refer to the
QuantiGene™ Plex Assay Kit Package Insert for individual component volumes or quantities.
Component
Proteinase K
Blocking ReagentAqueous buered solution containing a preservative-20°C
[1]
Validation Kit)
addition: Universal Platform Cat. No. 30100TS
and Universal Clamps for plates Cat. No. 30175)
DescriptionStorage
Proteinase K in aqueous buered solution-20°C
Label Probe SolutionBiotinylated oligonucleotide in aqueous buered
solution
Pre-Amplifier SolutionDNA in aqueous buered solution2-8°C
Amplifier SolutionDNA in aqueous buered solution2-8°C
SAPEStreptavidin-conjugated R-Phycoerythrin2-8°C
QuantiGene™ Plex Gene Expression Assay User Guide
2-8°C
7
Product information
Contents and storage
(continued)
ComponentDescriptionStorage
SAPE DiluentDilution Buer for SAPE reagent2-8°C
Lysis MixtureAqueous buered solution containing a preservative15-30°C
Pressure Seals (Day 1)Clear, pressure-activated seals for use with the
Magnetic Separation Plates96-well flat bottom microplates15-30°C
Plate Seals (Day 2)Clear, adhesive plate seals for use with the Magnetic
[1]
We recommend storing in an enzyme storage box, such as the NEB Cool Box (New England Biolabs P/N T0400S). NEVER store at
-80 °C.
QuantiGene™ Plex Panel
The QuantiGene™ Plex Panel includes the target-specific probe set and associated magnetic capture
beads. Each panel is supplied in 2 separate boxes based on storage temperature. Refer to the package
insert provided with the panel for the gene list and bead identifiers. Do not freeze the capture beads, as
they can be damaged if frozen.
Component
Probe setPre-mixed probe set consisting of target-specific capture
extenders, label extenders, and blocking probes
Capture beadsPre-mixed set of magnetic Luminex™ xMAP™ capture beads
conjugated with capture probes
15-30°C
Hybridization Plate during the Day 1/overnight
hybridization.
15-30°C
Separation Plate during the Day 2 hybridizations
DescriptionStorage
-20°C
2-8°C
8
QuantiGene™ Plex Gene Expression Assay User Guide
Before first use
•
Validate the magnetic plate washer to ensure proper bead retention. For instructions see
Appendix B, “Magnetic plate washer validation protocol”.
•
Calibrate the shaking incubator using the Temperature Validation Kit to ensure hybridization
temperatures are 54°C and 50°C. See instructions in the Temperature Validation Kit package insert.
•
Optimize sample preparation and input by running a dilution series to ensure all targets are
within the assay's dynamic range. For sample optimization instructions see “Sample optimization
protocol” on page 25.
Sample preparation
Prior to running the QuantiGene™ Plex Assay, ensure you have a lysate or homogenate prepared using
one of the following sample processing kits (of note, size/plate refers to 96-well plate format):
QS0104Fresh or Frozen Tissue Sample Processing Kit
QS0105Fresh or Frozen Tissue Sample Processing Kit25 samples
QS0106Fresh or Frozen Tissue Sample Processing Kit100 samples
QS0110Blood Sample Processing Kit
QS0111Blood Sample Processing Kit5 plates
QS0112Blood Sample Processing Kit5 × 10 Plate
QS0107FFPE Sample Processing Kit
QS0108FFPE Sample Processing Kit25 samples
QS0109FFPE Sample Processing Kit100 samples
[1]
Sufficient for preparing bulk lysates from 1.8 x 107 cells or 2 × 96-well plates containing up to 6 × 104 cells/well.
[2]
A sample is defined as 5 mg animal tissue or 15 mg plant tissue.
[3]
A 2-plate kit is sufficient for preparing bulk lysates from 1.8 × 107cells or 2 × 96-well plates containing up to 6 × 104cells/well.
[4]
A sample is defined as 25-100 mm2 × 50-60 microns (area × total thickness of FFPE tissue sections)
Assay specific reagentsSize
[1]
[2]
[3]
[4]
2 plate
10 samples
2 plates
10 samples
QuantiGene
™
Plex Gene Expression Assay User Guide
9
Assay procedure: day 1
IMPORTANT!
Tissue homogenates, cell lysates and whole blood lysates must be prepared using the applicable
·
QuantiGene™ Sample Processing Kit.
Purified RNA samples do not require a QuantiGene™ Sample Processing Kit.
·
The day 2 procedure is the same for all sample types.
·
For cell lysates or whole blood lysates
1.
Pre-warm Lysis Mixture at 37°C for 30 minutes followed by gentle swirling.
2.
If lysates have been frozen, remove from the freezer and thaw at room temperature followed by
incubation at 37°C for 30 min. Following incubation, vortex briefly if samples are in tubes or pipette
up and down 5 times if samples are in plates. Leave at room temperature until use.
Do not store on ice prior to use.
3.
Handle the reagents listed below as follows:
a.
Probe Set & Blocking Reagent: Thaw and vortex briefly to mix, then centrifuge Probe Set
briefly to collect contents at the bottom of the tube.
b.
Proteinase K: Keep on ice.
c.
Capture Beads: Take out of storage right before use and protect from light.
4.
If samples require dilution, dilute with Diluted Lysis Mixture (dilute 1 volume Lysis Mixture plus 2
volumes Nuclease-free Water, prepared fresh) so that the desired amount of sample is present in a
volume of 80 μL/assay well. In order to optimize sample input, please see “Optimize sample input”
on page 25.
10
QuantiGene™ Plex Gene Expression Assay User Guide
Assay procedure: day 1
For cell lysates or whole blood lysates
5.
Prepare an appropriate volume of Working Bead Mix by combining the following reagents in the
order listed. Scale according to the number of assays to be run, and include sucient overage.
Keep Working Bead Mix at room temperature and protected from light. Do not store on ice.
OrderReagent
1Nuclease-free Water5.26244. 2504
2Lysis Mixture6.67926.6792
3Blocking Reagent22402240
4Proteinase K0.2240.224
5Capture Beads
(vortex 30 secondsbefore adding)
6Probe Set56006720
Total202,400202,400
[1]
Includes 25% overage to enable use of reagent reservoir and multichannel pipette.
6.
Vortex Working Bead Mix for 10 seconds, then pipette 20 μL into each well of the Hybridization
1 Well (µL)96 Well (µL)
2 to 64-plex65 to 80-plex
[1]
11201120
1 Well (µL)96 Well (µL)
Plate.
•
For fewer than 48 wells: Dispense 20 μL of Working Bead Mix into each well of the
Hybridization Plate using a single channel pipette.
•
For 48 or more wells: Transfer Working Bead Mix to a 25-mL reagent reservoir using a single
channel pipette. Do not pour or reagent shortage will occur. Using a multichannel pipette
and new tips for each transfer, dispense 20 μL Working Bead Mix into each well of the
Hybridization Plate.
[1]
7.
Add 80 μL of lysate or diluted lysate to each well of the Hybridization Plate containing Working
Bead Mix. The total final volume in each well will be 100 μL. Load each sample using a new pipette
tip.
Background Controls: Add 80μL of Diluted Lysis Mixture (1 volume Lysis Mixture plus 2 volumes
Nuclease-free Water) to at least 3 wells containing Working Bead Mix.
8.
Seal the Hybridization Plate using a Pressure Seal: Remove the backing of the Pressure Seal,
center and place onto the Hybridization Plate. Using a soft- rubber roller, apply firm even pressure
across the seal. Ensure that the plate has been completely sealed.
Note: DO NOT use the Day 2 Plate Seal, otherwise evaporation may occur.
9.
Place the Hybridization Plate in the shaking incubator and incubate for 18-22 hours at 54°C ± 1°C
at 600 rpm. Ensure the incubator has been calibrated using the Temperature Validation Kit. If using
a VorTemp™ 56, ensure there is an inverted plate lid in place, as explained by the package insert.
10.
After incubation, proceed to “Assay procedure: day 2” on page 16.
QuantiGene™ Plex Gene Expression Assay User Guide
11
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